Toward a comprehensive modification landscape of yeast mitochondrial tRNAs using Nanopore direct RNA sequencing and Dihydrouridine sequencing

Saccharomyces cerevisiae is an invaluable model in the study of mitochondrial tRNA biology. Yet the positions of modified bases in all yeast mitochondrially-encoded tRNAs (mt-tRNAs) are still not fully mapped. We performed Nanopore direct RNA sequencing (DRS) on tRNAs from the crude mitochondrial fraction of yeast to map base modifications across all 24 mt-tRNA isoacceptors. Additionally, we developed a method to detect dihydrouridine sites in tRNAs, tD-seq, where chemical reduction of dihydrouridine causes disruptions to reverse transcription. We mapped dihydrouridine, pseudouridine, and N2-dimethylguanosine sites in mt-tRNAs using DRS, tD-seq, and knockouts of five conserved tRNA-modifying enzymes. Our results establish Dus1 and Dus2 as the enzymes responsible for D ₁₄ , D ₁₆ , D ₁₇ , D _17a , and D ₂₀ formation in S. cerevisiae mt-tRNAs, and revealed interactions between Dus1, Dus2, and Trm1-catalyzed modifications. We provide a comprehensive analysis of S. cerevisiae mt-tRNA base modifications, and identify novel modification “circuits” in yeast mt-tRNAs, in which the loss of a single enzyme’s activity can change modification levels at sites catalyzed by other enzymes. These findings expand our understanding of mt-tRNA base modifications and their interdependence, and advance opportunities for the yeast model for investigating defects in human mt-tRNA function.